LINC00941 promoted in vitro progression and glycolysis of laryngocarcinoma by upregulating PKM via activating the PI3K/AKT/mTOR signaling pathway

BackgroundLINC00941 has been proved to be related to various tumors, but its relationship with laryngocarcinoma remains vague.MethodsLINC00941 expression in laryngocarcinoma tumor and laryngocarcinoma cells was determined by real time‐quantitative polymerase chain reaction (RT‐qPCR). Besides, the five‐year survival of laryngocarcinoma patients with different LINC00941 expression was analyzed with Kaplan–Meier survival analysis, and the clinical characteristics of laryngocarcinoma patients were also recorded. After transfection, cell viability, cell proliferation, apoptosis, cell cycle, migration, and invasion were detected by cell counting kit‐8 (CCK‐8), colony formation, flow cytometry, cell scratch, and Transwell assays, respectively. Glycolysis was assessed by the colorimetric method. Expressions of proliferation‐associated proteins, migration‐associated proteins, glycolysis‐associated proteins, and phosphatidylinositol 3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathway‐associated proteins were detected by Western blot.ResultsIn laryngocarcinoma tumor tissues and cells, LINC00941 was highly expressed. High expression of LINC00941 decreased the 5‐year survival of laryngocarcinoma patients, and it was positively related to lymph node metastasis and clinical stages. LINC00941 overexpression decreased apoptosis but promoted cell viability, proliferation, cell‐cycle progression, migration, and invasion, and glucose consumption and lactate production in laryngocarcinoma cells. Moreover, LINC00941 overexpression elevated expressions of Ki‐67, PCNA, MMP2, N‐Cadherin, HK2, PFKFB4, and PKM, activated the PI3K/AKT/mTOR signal pathway but reduced E‐Cadherin expression, while LINC00941 silencing had the opposite effects. PKM overexpression reversed the effects of LINC00941 silencing on cellular and glycolytic phenotypes.ConclusionLINC00941 promoted in vitro progression and glycolysis of laryngocarcinoma cells by upregulating PKM via activating the PI3K/AKT/mTOR signaling pathway.     

Silencing of lncRNA MIR31HG promotes nasopharyngeal carcinoma cell proliferation and inhibits apoptosis through suppressing the PI3K/AKT signaling pathway

BackgroundMIR31HG has been affirmed to regulate the tumorigenesis of head–neck squamous cell carcinoma (HNSC). This study aims to reveal the function of MIR31HG in nasopharyngeal carcinoma (NPC), which falls into the category of HNSC.MethodsMIR31HG expression pattern in HNSC tissues was predicted by starBase. FISH and qRT‐PCR were employed to detect MIR31HG expression in NPC tissues and to analyze the association between MIR31HG and clinicopathological features. NPC cell viability, colony formation, and apoptosis were measured by MTT assay, colony formation assay, and flow cytometry. The expressions of protein kinase B (AKT), phosphorylated (p)‐AKT, phosphoinositide 3‐kinases (PI3K) and p‐PI3K in NPC cells were analyzed by Western blot. The correlation between MIR31HG expression and AKT1 mRNA expression was analyzed by The Cancer Genome Atlas and starBase.ResultsMIR31HG was highly expressed in HNSC tissues and NPC tissues. Meanwhile, the association between high MIR31HG expression and aggressive clinicopathological traits was significant in NPC patients at tumor stage III‐IV (T3‐T4) and in those with lymph node metastasis 1–2 (N1‐N2). Silencing of MIR31HG suppressed NPC cell viability and colony formation, promoted apoptosis, and decreased the expressions of p‐PI3K, and p‐AKT. 740Y‐P reversed the above effects of si‐MIR31HG on NPC cells. Besides, MIR31HG expression was positively correlated with AKT1 mRNA expression in HNSC patients.ConclusionMIR31HG silencing promotes NPC cell proliferation and inhibits apoptosis through suppressing the PI3K/AKT signaling pathway.     

siRNA诱导K562细胞凋亡的实验研究

目的　构建抗bcr/ablmRNA的小干扰RNA(smallinterferenceRNA ,siRNA)表达载体 ,转染K5 6 2细胞 ,检测诱导细胞凋亡的变化。方法参照siRNA模板设计原则 ,设计并合成两条siRNA模板序列 ,将其插入质粒pSilencer1.0 U6中得到重组子pBCR6 ,通过限制性酶切和测序鉴定 ,大量制备、纯化 ;以X tremeGENEQ2介导瞬时转染K5 6 2细胞 ,设置空载体作为对照。在转染后不同时间 ,利用原位缺口末端标记法 (TUNEL)、膜联蛋白Ⅴ 碘化丙锭染色法 (AnnexinⅤ /PI)通过流式细胞仪检测K5 6 2细胞凋亡的变化。结果针对bcr/ablmRNA融合区域设计的siRNA模板序列 ,经筛选合成寡核苷酸链后退火形成双链 ,再插入pSilencer1.0 U6 ,经酶切和测序鉴定提示构建成功 ;大量制备、纯化后进行转染 ,在转染后 4 8,72hTUNEL法检测和AnnexinⅤ /PI染色法均显示抗bcr/ablmRNA的siRNA表达载体可有效诱导K5 6 2细胞凋亡 ,且随转染时间的延长凋亡率增高 [转染pBCR6 72h的K5 6 2细胞凋亡率为 (47.80± 1.6 3) % ],与对照组 [(6 .6 7± 0 .37) % ]比较差异有显著性 (P <0 .0 0 0 1)。结论抗bcr/ablmRNA的siRNA表达载体构建成功 ,初步结果显示它可以有效地诱导K5 6 2细胞发生凋亡 ,预期siRNA有望成为慢性髓系白血病分子靶向治疗的一个新工具。     

木香烃内酯通过PI3K/AKT通路诱导K562/ADR细胞凋亡

目的:探讨木香烃内酯对慢性粒细胞性白血病细胞耐药细胞株K562/ADR细胞增殖及凋亡的影响及其机制.方法:运用CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测相关蛋白的表达.结果:选用木香烃内醋0.01、0.1、0.25、0.5、1、2.5、5、10、25、50和100 μmol/L作用K...     

靶向siRNA沉默mdr1基因表达及对白血病耐药细胞系K562/ADM的耐药逆转作用

目的 研究小干扰RNA（siRNA）对白血病多药耐药细胞系K562／ADM细胞mdr1基因表达的沉默作用和凋亡抑制的逆转效应。方法 K562／ADM为靶细胞，设计、筛选和合成2对针对mdr1基因mRNA的siRNA（mdr1 siRNA-1和mdr1siRNA-2），用脂质体介导转染K562／ADM细胞；实时荧光定量PCR（real—time PCR）法检测mdr1 mRNA的表达；流式细胞术测定P-糖蛋白（P—gP）水平和caspase-3活性；细胞形态学和FITC标记的膜联蛋白V／碘化丙锭（Annexin V—FITC／PI）双染色法检测细胞的凋亡。结果 筛选出的mdr1 siRNA-1和mdr1 siRNA-2显著抑制K562／ADM细胞mdr1的表达，mdr1 mRNA的表达分别降低91．2％和82．0％，P-gp水平下降74．1％和84．4％；增强caspase-3活性，活化caspase-3增加约40％；K562／ADM耐药细胞对阿霉素诱导凋亡的敏感性增强，Annexin V—FITC／PI染色检测细胞凋亡率提高约60％。结论 siRNA通过沉默mdr1 ／P—gp表达而逆转K562／ADM多药耐药细胞的凋亡抑制现象。     

槲皮素逆转白血病细胞系K562/A多药耐药性及相关基因表达的研究

本课题研究槲皮素(quercetin,Que)对白血病细胞系K562/A多药耐药的影响及机制.体外培养的K562和K562/A细胞经不同浓度的Que处理,采用MTT法检测Que对细胞的生长抑制率及对阿霉紊(adriamycin,ADR)的增敏倍数;流式细胞术检测Que作用后细胞内ADR浓度的变化,Annexin V/PI双染色法观察细胞凋亡情况;实时荧光定量PCR基因芯片检测药物转运蛋白基因及凋亡相关基因表达的变化.结果表明,Que在5-160μmol/L的浓度范围内对K562和K562/A细胞均有剂量依赖性的生长抑制作用,低毒剂量的Que使K562/A对ADR的敏感性显著增强;在ADR为5 μmol/L时,Que与细胞共培养2小时细胞内ADR浓度则明显增加;Que可剂量依赖性地诱导K562和K562/A细胞的凋亡;Que可下调ABC、SLC家族药物转运蛋白相关基因的表达,并可调节BCL-2、TNF等凋亡相关基因的表达.结论:Que可通过多种机制逆转白血病细胞系K562/A的多药耐药性,逆转效果与剂量呈正相关.     

WAVE1基因在K562/A02白血病细胞多药耐药中的作用

目的 探讨WASP家族Verprolin同源蛋白1（WAVE1）基因在K562／A02白血病细胞多药耐药机制中的作用。方法 将pEFBOS—WAVE1真核表达质粒转染K562细胞构建WAVE1高表达的K562细胞，将WAVE1基因的特异性小片段干扰RNA（WAVE1siRNA）转染K562／A02细胞构建WAVE1低表达的K562／A02细胞。Western blot和RT-PCR法检测基因转染前后K562／A02细胞及K562细胞WAVE1基因及蛋白的表达；可溶性噻唑盐WST-8染色法检测阿霉素对转染前后细胞的半数抑制浓度（IC50）；Hoechest33258染色法检测细胞形态学改变并计算凋亡细胞百分率；RT—PCR检测多药耐药基因mdrl mRNA表达；Western blot检测Bcl-2表达。结果 ①与K562细胞相比，K562／A02细胞WAVE1 mRNA表达水平增加70％，蛋白表达水平增加63％。②转染WAVE1基因的K562细胞WAVE1过表达，并降低了对阿霉素的药物敏感性，使IC50从转染前的（0．05±0．00）μg／ml，增加到（2．99±0．12）μg／ml，在阿霉素浓度为1.5μg／ml时，凋亡细胞分别下降30％、35％。③转染WAVE1 siRNA的K562／A02细胞WAVE1蛋白表达水平与未转染组相比明显降低，并可增强对阿霉素的药物敏感性，使IC50从转染前的（4．29±0．15）μg／ml下降到（1．85±0．07）μg／ml，阿霉素浓度为1.5μg／ml时，凋亡细胞分别增加24％、21％。④转染WAVE1的K562细胞mdr1基因和Bcl-2蛋白表达水平均增高，而转染WAVE1 siRNA的K562／A02细胞mdr1基因和Bcl-2蛋白表达水平比转染前明显降低。结论 WAVE1参与了K562／A02细胞多药耐药的形成，其机制可能与调控mdr1和Bcl-2水平有关。     

8-溴-7-甲氧基白杨素诱导K562细胞凋亡作用研究

本研究探讨8-溴-7-甲氧基白杨素(8-bromo-7-methoxyhysin,BrMChR)对人髓系白血病细胞株K562细胞增殖和凋亡的影响及细胞凋亡过程中caspase-3活性与p-Akt蛋白表达的变化。采用MTT方法检测BrMChR对K562细胞生长抑制的影响,用细胞形态学观察和流式细胞术分析细胞凋亡,Western blot方法检测p-Akt蛋白的表达。结果显示:与同浓度的白杨素(chrysin,ChR)相比,BrMChR对K562细胞的生长抑制作用及凋亡诱导作用更强,并随药物剂量的增加而加强;3μmol/L BrMChR作用K562细胞12小时细胞凋亡率可达21.8%,而同浓度的ChR作用K562细胞12小时细胞凋亡率仅为3.68%。随着BrMChR浓度增加,caspase-3活性增加(p<0.05),p-Akt蛋白表达下调(p<0.01)。结论 :BrMChR能诱导K562细胞凋亡,其作用强于ChR;p-Akt可能参与了BrMChR诱导K562细胞凋亡过程。     

核糖体蛋白L6对K562/A02细胞耐药性及凋亡的影响

本研究观察核糖体蛋白L6（RPL6）基因表达的改变对白血病细胞耐药性的作用及其可能的机制。通过RT-PCR方法获得RPL6 cDNA序列,用真核表达载体pcDNA3.1（＋）分别构建正向插入和反向插入的RPL6 cDNA重组质粒。以脂质体将正义RPL6 cDNA真核表达质粒转染K562细胞,将反义RPL6 cDNA真核表达质粒转染K562/AO2细胞。以MTT、流式细胞术和荧光分光光度计观察RPL6对化疗药物耐药性、凋亡和caspase-3的作用。结果表明：转染正义RPL6 cDNA真核表达质粒后,K562细胞对阿霉素的耐药性增强到原来的325%,凋亡和caspase-3活性明显降低（P〈0.005）;转染反义RPL6 cDNA真核表达质粒后,K562/A02细胞对阿霉素的耐药性降低原来的38%;凋亡和caspase-3活性明显增加（P〈0.005）。结论：RPL6基因过表达通过改变药物诱导的凋亡在K562/A02细胞耐药性的形成中起重要作用。     

亚硒酸钠对K562/ADR细胞系VEGF表达的影响

为了探讨亚硒酸钠对K562/ADR细胞VEGF表达的影响,分别以5、10μmol/L的亚硒酸钠对培养的K562及K562/ADR细胞进行处理,应用ELISA法检测亚硒酸钠处理前和处理后不同时间的K562及K562/ADR细胞上清液中VEGF含量,用MTT法检测逆转耐药倍数。结果表明：亚硒酸钠可以增加K562/ADR细胞对阿霉素的敏感性,其逆转耐药的倍数为3.48倍;两种细胞系分泌的VEGF含量随培养时间的延长增加,并且各个时间段的K562/ADR细胞VEGF表达均高于K562细胞（P〈0.05）;5、10μmol/L的亚硒酸钠在72小时内均不能抑制K562细胞VEGF的分泌（P〉0.05）;而作用96小时时K562细胞上清液中VEGF水平虽有下降,但未达统计学意义;5、10μmol/L的亚硒酸钠在48小时内均不能抑制K562/ADR细胞VEGF的分泌（P〉0.05）,10μmol/L的亚硒酸钠在作用72和96小时可以明显抑制VEGF的分泌（P〈0.001）,5μmol/L的亚硒酸钠在作用96小时可以抑制VEGF的分泌（P〈0.001）。结论：VEGF可能与白血病多药耐药有关,而亚硒酸钠则能抑制白血病细胞分泌VEGF。     

siRNA逆转K562/A02细胞多药耐药的研究

目的　研究小分子干扰RNA片段 (smallinterferingRNAs,siRNA)对白血病多药耐药细胞系K5 6 2 A0 2mdr1基因和P 糖蛋白 (P gp)表达及功能的影响。方法　根据mdr1基因已知序列设计 3条含 2 1个碱基的siRNA(si mdr1 1,si mdr1 2 ,si mdr1 3)及阴性对照 (si neg) ,在脂质体的介导下转染K5 6 2 A0 2细胞 ;用RT PCR分析mdr1mRNA的表达 ;流式细胞术检测P gp表达和细胞内柔红霉素积累量 ;MTT法检测阿霉素对K5 6 2 A0 2细胞的半数抑制浓度 (IC50 )。结果　 3条siRNA均能不同程度地逆转K5 6 2 A0 2细胞的多药耐药。第 3条序列能更有效地封闭mdr1基因 ,使mdr1mRNA相对水平下降(5 8.0± 1 5 4 ) % ;P gp表达由处理前的 (76 .0± 1.0 ) %降到处理后的 (19.6± 1.9) % ;对阿霉素药物敏感性的相对逆转效率为 70 .4 % ;同时使K5 6 2 A0 2细胞内柔红霉素积累量增加。结论　siRNA可逆转mdr1基因编码蛋白P gp介导的多药耐药。     

K562细胞血管内皮生长因子自分泌链作用的实验研究

目的探讨抗VEGF抗体及抗VEGF发夹状核酶基因对K562细胞增殖、凋亡及相关基因表达的影响及其分子机制。方法将不同浓度的VEGF抗体作用于K562细胞，应用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体pcDNA-RZ转染K562细胞，采用MTT法、甲基纤维素半固体培养法和细胞周期测定分析抗VEGF抗体及发夹状核酶基因对白血病细胞增殖的影响；DNA凝胶电泳和AnnexinⅤ标记法检测白血病细胞的凋亡程度；RT-PCR法测定K562细胞中相关基因表达的变化。结果抗VEGF抗体能抑制K562细胞生长，促进其凋亡，这一作用呈剂量依赖关系。0．165μg／ml VEGF抗体作用于K562细胞72h，DNA凝胶电泳出现梯状条带，当抗体浓度增加到0．825μg／ml时，梯状条带更为清晰；RT-PCR显示，VEGF抗体作用后，K562细胞MRP、TOPOⅡ基因的表达较对照组下调，而GST基因表达无明显改变。与K562及K562／PC细胞（转染空质粒的K562细胞）相比，转染抗VEGF核酶基因的K562／RZ细胞VEGF mRNA和蛋白的表达量明显降低；生长曲线提示K562／RZ细胞生长缓慢，甲基纤维素集落形成率较对照组明显下降，对照组集落形成率为（30．5±3．3）％，而K562／RZ组为（16．3±2．8）％，细胞周期动力学分析结果显示K562／RZ细胞G1期细胞增多，S期细胞显著减少；在小剂量As2O3作用下，K562／RZ细胞凋亡数量较对照明显增高（凋亡率对照组为13．4％，K562／RZ组为31．5％）。结论抗VEGF抗体阻断K562细胞VEGF自分泌环路或者抗VEGF发夹状核酶减少K562细胞中VEGF的合成，能抑制K562细胞的增殖，促进K562细胞的凋亡，引起部分与细胞凋亡和多药耐药相关的基因表达改变。     

亚硒酸钠对K562/ADR多药耐药的逆转作用及其机制

本研究探讨亚硒酸钠对白血病耐药细胞株K562/ADR细胞的多药耐药性的逆转作用及其逆转机制。用噻唑蓝（MTT）法检测亚硒酸钠对K562/ADR细胞的生长抑制作用及其对K562/ADR细胞耐药性的逆转作用;应用流式细胞仪检测亚硒酸钠对K562及K562/ADR细胞凋亡率的影响;用半定量RT-PCR法检测K562/ADR细胞中mdr1和bcl-2基因的表达情况。结果表明,10μmol/L亚硒酸钠可以明显增加K562/ADR细胞对阿霉素敏感性,其耐药逆转倍数为2.31;早期凋亡率在10μmol/L亚硒酸钠作用于K562细胞48小时是升高的;而中、晚期凋亡率在亚硒酸钠5、10μmol/L作用48、72小时时均明显升高;两种浓度亚硒酸钠在作用48小时可使K562/ADR细胞早期凋亡率增加,作用48、72小时可使K562/ADR的细胞中、晚期凋亡率增高,10μmol/L的亚硒酸钠所致凋亡率高于5μmol/L,作用72小时的凋亡率高于48小时;亚硒酸钠可使K562/ADR细胞mdr1 mRNA及bcl-2mRNA表达下降。结论：亚硒酸钠可部分逆转K562/ADR细胞的耐药性,其机制可能是增加K562/ADR细胞凋亡率,下调mdr1 mR-NA和bcl-2 mRNA的表达。     

HMGB1基因沉默对阿霉素诱导K562/A02细胞凋亡增敏作用的研究

目的 探讨高迁移率族蛋白1(HMGB1)基因沉默对白血病细胞耐药逆转的作用.方法 将HMGB1基因特异性干扰RNA(HMGB1 siRNA)导入K562/A02细胞中,通过Western blot和RTPCR方法检测HMGB1基因在转染前后的表达;WST8法检测阿霉素(ADM)对转染前后K562/A02细胞的半数抑制浓度(IC50);流式细胞术检测凋亡细胞百分率;Western blot检测线粒体促凋亡蛋白Smac/DIABLO的释放;采用caspase活性定量检测试剂盒分析caspase-3的活性.结果 ①与未经处理的K562/A02细胞组相比,转染HMGB1 siRNA的K562/A02细胞组HMGB1 mRNA和蛋白水平分别下降86%和71%;②HMGB1基因沉默使K562/A02细胞对ADM的药物敏感性增强,其IC50值从转染前的(4.83±0.08)μg/ml降低到(1.33±0.10)μg/ml,并在ADM浓度为1μg/ml和5μg/ml时,细胞凋亡百分率分别增加27%、32%;③HMGB1基因沉默可促进ADM所致Smac/DIABLO从线粒体向胞浆释放,并增加caspase-3的活性.结论 HMGB1基因沉默能明显增加K562/A02细胞对ADM的敏感性,逆转K562/A02细胞对ADM耐药.     

Inhibition of the Akt/survivin pathway synergizes the antileukemia effect of nutlin-3 in acute lymphoblastic leukemia cells

The phosphatidylinositol 3-kinase (PI3K)/Akt and p53 pathways play antiapoptotic and proapoptotic roles in cell death, respectively. Cancer cell growth and progression are associated with high levels of PI3K/Akt activation by loss of PTEN expression and the inactivation of p53 by MDM2 overexpression. We report that inhibition of PI3K/Akt, either by the PI3K inhibitor Ly294002 or by expression of PTEN, synergized the ability of the MDM2 antagonist nutlin-3 to induce apoptosis in acute lymphoblastic leukemia (ALL). We used a set of ALL cell lines with wild-type p53 and MDM2 overexpression, but different status of PTEN expression/PI3K/Akt activation, to test the ability of nutlin-3 to induce p53 and apoptosis. Nutlin-3 activated p53 in all the ALL cell lines; however, induction of apoptosis was dependent on PTEN status. Nutlin-3 induced potent apoptosis in cells with PTEN expression but not in those without PTEN, suggesting that PTEN/PI3K/Akt pathway may play a role in this process. Furthermore, nutlin-3 significantly down-regulated survivin expression in PTEN-positive cells but not in PTEN-negative cells. When these nutlin-3-resistant cells were either pretransfected with the PTEN gene or simultaneously treated with the PI3K inhibitor Ly294002, survivin was down-regulated and sensitivity to nutlin-3 was increased. Furthermore, direct silencing of survivin by small interfering RNA also increased the proapoptotic effect of nutlin-3 on the PTEN-negative, nutlin-3-resistant ALL cells. Our results suggest that Akt-mediated survivin up-regulation in PTEN-negative ALL cells may counteract the proapoptotic effect of nutlin-3, and indicate that a combination of MDM2 antagonist and PI3K/Akt inhibitor may be a promising approach for treating refractory ALL.     

汉防己甲素、托瑞米芬及其联合应用逆转K562/A02细胞多药耐药的研究

本研究探讨汉防己甲素（Tet）、雌激素受体抑制剂托瑞米芬（Tor）及其联合应用对K562/A02细胞多药耐药的逆转作用。采用噻唑蓝法（MTT）测定阿霉素（ADR）的半数抑制量,RT-PCR法检测MDR1基因mRNA表达水平,FCM检测P-gp的表达和细胞内ADR浓度。结果表明：ADR对K562/A02和K562细胞的IC50分别为57.43和1.16mg/L,Tet（1μmol/L）和Tor（2.5μmol/L）单用及两药联用处理K562/A02细胞时,ADR的IC50值分别为14.12,20.74和9.14mg/L。Tet及Tor单独作用后耐药株MDR1 mRNA下调微弱,联合用药下调效果明显。Tet及Tor均可降低P-gp蛋白的表达,且具有协同作用。Tet及Tor作用后细胞内ADR浓度增加,两药联合作用时效果增强。结论：Tet及Tor均可逆转K562/A02细胞多药耐药,联合作用时效果增强。     

AG490抑制K562细胞增殖和下调PHLPP蛋白的表达

本研究旨在探讨AG490抑制K562细胞增殖并诱导其凋亡过程中p-Akt及PHLPP表达的变化。以不同浓度的AG490作用于人慢性髓系急性红白血病细胞株K562,采用WST-1法检测细胞的增殖活性,AnnexinⅤ-FITC双染检测细胞凋亡,Western blot检测p-Akt、Akt、PHLPP蛋白表达水平的变化。结果表明,AG490以时间及浓度依赖性方式抑制K562细胞增殖,其48 h的IC50值为338.0μmol/L;AG490 100μmol/L诱导K562细胞凋亡,呈明显的时间依赖性;AG490 100μmol/L作用于K562细胞后p-Akt及其调节蛋白PHLPP的表达水平呈时间依赖性方式下降,但是对总Akt的表达水平无明显影响。结论:AG490可能通过下调p-Akt的表达抑制K562细胞增殖并诱导其凋亡,但同时也可能通过下调p-Akt的调节蛋白PHLPP的表达水平而使AG490对K562增殖的抑制效果受限。     

CIK逆转K562/ADR细胞多药耐药作用及其机制探讨

目的 研究细胞因子诱导的杀伤细胞(CIK)体外逆转阿霉素(ADR)耐药细胞株K562/ADR细胞多药耐药(MDR)的作用,并探讨其机制.方法 健康人外周血单个核细胞经细胞因子体外诱导获得CIK,检测其表型和培养上清液细胞因子含量.实验组为CIK作用于K562/ADR细胞48 h后加入ADR;对照1组为CIK作用于K562/ADR细胞48 h,对照2组为ADR作用K562/ADR细胞48 h.采用MTT法检测各组细胞杀伤活性,用流式细胞术检测细胞膜P-糖蛋白(P-gp)含量、细胞内ADR浓度等.结果 实验组对K562/ADR细胞杀伤活性高于对照1组(P＜0.05);且随效靶比增大,杀伤活性增大(P＜0.05);随所加入的ADR浓度增大,杀伤活性无明显变化(P＞0.05).实验组和对照1组的P-gp含量均下降(P＞0.05).实验组细胞内ADR浓度高于仅经ADR作用的对照组2组(P＜0.05),但细胞内ADR浓度与加入的ADR浓度无明显关系(P＞0.05).结论 通过CIK与ADR对K562/ADR细胞的先后作用,降低了其细胞内P-gp的表达,提高了K562/ADR细胞内ADR浓度,增强了ADR对耐药细胞的杀伤活性.为应用生物活性细胞逆转MDR提供理论依据.

Abstract：

Objective To investigate the effects and mechanism of cytokine-induced killer(CIK) cells in reversing multidrug resistance(MDR) and increasing intracellular concentration of adriamycin(ADR)in the K562/ADR cells. Methods Peripheral mononuclear cells (MNCs) were isolated from healthy donors and cultured with combined cytokines to generate CIK. The changes of cell phenotype and cytokines secretion of CIK were determined. K562/ADR cells were divided into three groups: ADR in combination CIK (group Ⅰ ), CIK alone (group Ⅱ ) and ADR alone (groupⅢ) . The viability and proliferation of K562/ADR cells were assayed by MTT assay, the intracellular concentration of ADR and the expression of P-glycoproteins (P-gp) in K562/ADR cells by FCM. Results The cytotoxicity of ADR in group Ⅰ was higher than that in group Ⅱ ( P ＜0.05 ). The cytotoxicity was increased with the E/T ratio increasing( P ＜0.05 ) but had no relation with the concentration of ADR in group Ⅰ (P＞0.05). The expression of P-gp was declined in group Ⅰ and group Ⅱ (P ＞0.05 ). The intracellular concentration of ADR in group Ⅰ was higher than that in group Ⅱ ( P ＜ 0.05 ), and had no relation with the ADR concentration ( P ＞ 0.05 ). Conclusion Pre-treatment with CIK can increase the cytotoxicity and the intracellular concentration of ADR and decrease the expression of P-gp in K562/ADR cells in the ADR and CIK combination group. Acute leukemia patients would be most likely to benefit from the combination of chemotherapy and CIK therapy.     

舒尼替尼对白血病细胞K562作用及其分子机制研究

本研究旨在探讨舒尼替尼对白血病K562细胞的作用及其机制.应用MTT法检测以IC50为3.2μg/ml的舒尼替尼作用不同时间后K562细胞的增殖情况;用凝胶电泳分析DNA片段化、用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测舒尼替尼对K562细胞凋亡的影响;应用RT-PCR检测3.2 μg/ml舒尼替尼作用于K562细胞后C-MYC、hTERT、BCR-ABL mRNA的表达变化;Western blot检测不同浓度舒尼替尼处理K562细胞后和3.2μg/ml舒尼替尼作用不同时间后Akt、p-Akt蛋白表达的变化.结果表明,舒尼替尼可明显抑制K562细胞增殖,呈时间依赖性;舒尼替尼诱导K562细胞呈现典型的DNA梯状带,促进细胞原位凋亡;3.2μg/ml舒尼替尼作用后K562细胞C-MYC、hTERT、BCR-ABL mRNA表达呈时间依赖性降低;Western blot显示,p-Akt蛋白呈时间和剂量依赖性降低,而Akt蛋白表达无明显变化.结论：舒尼替尼能有效地通过诱导凋亡抑制K562细胞增殖,其机制可能与下调BCR-ABL、C-MYC和hTERT的表达及抑制Akt磷酸化有一定的关系.